Proteins fold to adopt unique three dimensional structures, usually as a result of multiple non-covalent interactions that contribute to their conformational stability. Creighton, T. E. Proteins: Structures and Molecular Properties; 2nd ed.; W. H. Freeman: New York, 1993. Removal of hydrophobic surface area from aqueous solvent plays a dominant role in stabilizing protein structures. Tanford, C. Science 1978, 200, 1012–1018; and Kauzmann, W. Adv. Protein Chem. 1959, 14, 1–63. For instance, a buried leucine or phenylalanine residue can contribute ˜2–5 kcal/mol in stability when compared to alanine. Although hydrogen bonds and salt bridges, when present in hydrophobic environments, can contribute as much as 3 kcal/mol to protein stability, solvent exposed electrostatic interactions contribute far less, usually 0.5 kcal/mol. Yu, Y. H.; Monera, O. D.; Hodges, R. S.; Privalov, P. L. J. Mol. Biol. 1996, 255, 367–372; and Lumb, K. J.; Kim, P. S. Science 1995, 268, 436–439. Hydrogen bonds between small polar side chains and backbone amides can be worth 1–2 kcal/mol, as seen in the case of N-terminal helical caps. Aurora, R.; Rose, G. D. Protein Sci. 1998, 7, 21–38. The energetic balance of these intramolecular forces and interactions with the solvent determines the shape and the stability of the fold.
While electrostatic interactions in designed structures can provide conformational specificity at the expense of thermodynamic stability, hydrophobic interactions afford a very powerful driving force for stabilizing structures. Recent studies have focused on the introduction of non-proteinogenic, fluorine containing amino acids as a means for increasing hydrophobicity, without significant concurrent alteration of protein structure. Bilgicer, B.; Fichera, A.; Kumar, K. J. Am. Chem. Soc. 2001, 123, 4393–4399; and Tang, Y.; Ghirlanda, G.; Vaidehi, N.; Kua, J.; Mainz, D. T.; Goddard, W. A.; DeGrado, W. F.; Tirrell, D. A. Biochemistry 2001, 40, 2790–2796. The estimated average volumes of CH2 and CH3 groups are 27 and 54 Å3, respectively, as compared to the much larger 38 and 92 Å3 for CF2 and CF3 groups. Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Biochim. Biophysica Acta 1977, 470, 185–201. Given that the hydrophobic effect is roughly proportional to the solvent exposed surface area, the large size and volume of trifluoromethyl groups, in combination with the low polarizability of fluorine atoms, results in enhanced hydrophobicity. Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes; 2d ed.; Wiley: New York, 1980. Indeed, partition coefficients point to the superior hydrophobicity of CF3 (Π=1.07) over CH3 (Π=0.50) groups. Resnati, G. Tetrahedron 1993, 49, 9385–9445. The low polarizability of fluorine also results in low cohesive energy densities of liquid fluorocarbons and is manifested in their low propensities for intermolecular interactions. Riess, J. G. Colloid Surf.-A 1994, 84, 33–48; and Scott, R. L. J. Am. Chem. Soc. 1948, 70, 4090–4093. These unique properties of fluorine simultaneously bestow hydrophobic and lipophobic character to biopolymers with high fluorine content. Marsh, E. N. G. Chem. Biol. 2000, 7, R153–R157.
Introduction of amino acids containing terminal trifluoromethyl groups at appropriate positions on protein folds increases the thermal stability and enhances resistance to chemical denaturants. Bilgicer, B.; Fichera, A.; Kumar, K. J. Am. Chem. Soc. 2001, 123, 4393–4399; and Tang, Y.; Ghirlanda, G.; Vaidehi, N.; Kua, J.; Mainz, D. T.; Goddard, W. A.; DeGrado, W. F.; Tirrell, D. A. Biochemistry 2001, 40, 2790–2796. Furthermore, specific protein-protein interactions can be programmed by the use of fluorocarbon and hydrocarbon side chains. Bilgicer, B.; Xing, X.; Kumar, K. J. Am. Chem. Soc. 2001, 123, 11815–11816. Because specificity is determined by the thermodynamic stability of all possible protein-protein interactions, a detailed fundamental understanding of the various combinations is essential.
The so-called “leucine zipper” protein motif, originally discovered in DNA-binding proteins but also found in protein-binding proteins, consists of a set of four or five consecutive leucine residues repeated every seven amino acids in the primary sequence of a protein. In a helical configuration, a protein containing a leucine zipper motif presents a line of leucines on one side of the helix. With two such helixes alongside each other, the arrays of leucines can interdigitate like a zipper and/or form side-to-side contacts, thus forming a stable link between the two helices. Moreover, an increase in the hydrophobicity of the leucine sidechains, e.g., by substitution of hydrogens with fluorines, in a leucine zipper motif should increase the strength of the zipper.
Selective fluorination of biologically active compounds is often accompanied by dramatic changes in physiological activities. (a) Welch, T.; Eswarakrishnan, S. Fluorine in Bioorganic Chemistry; Wiley-Interscience: New York, 1991 and references cited therein; (b) Fluorine-containing Amino Acids; Kukhar', V. P., Soloshonok, V. A., Eds.; John Wiley & Sons: Chichester, 1994; (c) Williams, R. M. Synthesis of Optically Active α-Amino Acids, Pergamon Press: Oxford, 1989; (d) Ojima, I.; Kato, K.; Nakahashi, K.; Fuchikami, T.; Fujita, M. J. Org. Chem. 1989, 54, 4511–4522; (e) Tsushima, T.; Kawada, K.; Ishihara, S.; Uchida, N.; Shiratori, O.; Higaki, J.; Hirata, M. Tetrahedron 1988, 44, 5375–5387; (f) Weinges, K.; Kromm, E. Liebigs Ann. Chem. 1985, 90–102; (g) Eberle, M. K.; Keese, R.; Stoeckli-Evans, H. Helv. Chim. Acta 1998, 81, 182–186; and (h) Tolman, V. Amino Acids 1996, 11, 15–36. Further, fluorinated amino acids have been synthesized and studied as potential inhibitors of enzymes and as therapeutic agents. Kollonitsch, J.; Patchett A. A.; Marburg, S.; Maycock, A. L.; Perkins, L. M.; Doldouras, G. A.; Duggan, D. E.; Aster, S. D. Nature 1978, 274, 906–908. Trifluoromethyl containing amino acids acting as potential antimetabolites have also been reported. (a) Walborsky, H. M.; Baum, M. E. J. Am. Chem. Soc. 1958, 80, 187–192; (b) Walborsky, H. M.; Baum, M.; Loncrini, D. F. J. Am. Chem. Soc. 1955, 77, 3637–3640; and (c) Hill, H. M.; Towne, E. B.; Dickey, J. B. J. Am. Chem. Soc. 1950, 72, 3289–3289.
We describe herein inter alia the design, synthesis, thermodynamic characterization and programmed self-sorting of peptide systems with orthogonally miscible hydrocarbon and fluorous, i.e., highly fluorinated cores.